T cell activation markers

ABSTRACT

A polypeptide designated H400 and its encoding nucleic acid are provided as markers specific for activated human T cells. Activated t cells are detected immunochemically by monoclonal antibodies specific for H400 or its immunogenic peptides. Activated T cells are also detected by nucleic acid probes directed to messenger RNA encoding the H400 protein.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions formonitoring the status of the immune system; more particularly, theinvention provides compositions for detecting activated T-cells bynucleic acid hybridization probes and/or immunochemical assays.

BACKGROUND

Regulation of normal immunologic reactivity involves the balance betweenpositive and negative influences exerted by various subsets of T cells.Abnormally hyperactive and hypoactive T cells are believed to beassociated with several immune disorders, including AIDS (hypoactivityand destruction of the helper T cell subset), and a variety ofautoimmune disorders, such as MHC-associated autoimmune disease (e.g.lupus erythematosus) which is believed to be caused by excessiveproduction of gamma interferon by T cells.

T cells have been identified by certain phenotypic cell surface markers.For example, the surface molecule T8, which is present on human T cellsof the cytotoxic subset, can be detected by immunofluorescent stainingusing murine anti-human monoclonal antibodies, such as OKT8 (OrthoDiagnostics, Raritan, NJ) or Leu-2 (Becton-Dickinson, Mountain View,CA). However, such markers are, at best, only useful in measuring thesize of subpopulations, they do not necessarily imply that the cells aresecreting products associated with an activated state.

Many diagnostic assays have been developed which are based on eitherimmunochemical detection of proteins, e.g. U.S. Pat. Nos. 4,562,003;4,474,892; and 4,427,782; or the detection of nucleic acids, either RNAor DNA, present in or produced by target cells, e.g. Pettersson et al.,Immunology Today, Vol. 6, pgs. 268-272 (1985); Falkow et al., U.S. Pat.No. 4,358,535; and Gillespie et al., U.S. Pat. No. 4,483,920. The latterassays use nucleic acid probes, usually fluorescently labeled orradioactively labeled DNAs or RNAs, which can be preferentiallyhybridized to complementary target nucleic acids in appropriatelyprepared samples or tissues. The assays can take a variety of forms,e.g. RNA blotting: Thomas, Proc. Natl. Acad. Sci., Vol. 77, pgs.5201-5205 (1980); dot hybridization: White et al., J. Biol. Chem., Vol.257, pgs. 8569-8572 (1982); Southern blotting: Southern, J. Mol. Biol.,Vol. 98, pgs. 503-517; and in situ hybridization: Pinkel et al., Proc.Natl. Acad. Sci., Vol. 83, pgs. 2934-2938 (1986); and Angerer et al.,Genetic Engineering, Vol. 7, pgs. 43-65 (1985).

In view of the present lack of convenient direct methods for measuringabnormal T cell activation, the availability of sensitive immunochemicalassays or nucleic acid probes for detecting activated T cells wouldprovide useful diagnostic tools.

SUMMARY OF THE INVENTION

The invention includes a mature human protein produced by activated Tcells, designated herein as H400, and immunogenic peptides thereof, bothof which are useful for generating antibodies employed in immunochemicalassays for activated T cells. The amino acid sequence corresponding tothe open reading frame encoding H400 is given in Formula I. ##STR1## Theinvention further includes monoclonal antibodies specific for the matureH400 protein and its immunogenic peptides, and nucleic acids encodingH400 and fragments thereof useful in the construction of nucleic acidprobes for H400 messenger RNA (mRNA). The preferred nucleotide sequenceof the open reading frame encoding H400 is given in Formula II.

A plasmid, designated herein as pcD(SRα)-H400, containing a cDNA insertcapable of encoding H400 has been deposited with the American TypeCulture Collection (Rockville, MD) under accession number 67614.##STR2##

As used herein, the term "mature" in reference to protein H400 means thesecreted form of H400. That is, the form of H400 that results fromproteolytic cleavage of the signal peptide.

As used herein, "immunogenic peptide" in reference to H400 means apeptide (1) which consists of from 6 to 40 amino acids having a sequenceidentical to an equal length portion of mature H400 and (2) which inconjugation with a carrier protein is capable of eliciting antibodieswhich cross react with mature H400.

As used herein, "nucleic acid fragment" means a nucleic acid whichconsists of from about 18 to 264 nucleotides having a sequencecomplementary to an equal length portion of the nucleic acid defined byFormula II.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrammatically illustrates the plasmid pcD(SRα)-H400 andselected restriction sites.

FIG. 2 lists the nucleotide sequence of the cDNA insert of pcD(SRα)-H400and indicates the amino acid sequence corresponding to the largest openreading frame.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery of a novel protein, H400, whichis produced specifically by activated T cells. The protein H400 isencoded by the largest open reading frame of the cDNA insert ofpcD(SRα)-H400. The invention is directed to compounds useful indetecting cells which either produce the H400 protein or produce mRNAtranscripts capable of encoding the H400 protein. These compoundsinclude the mature protein H400, immunogenic peptides thereof,monoclonal antibodies specific for mature H400 or its immunogenicpeptides, nucleic acids capable of encoding H400, and nucleic acidfragments for constructing nucleic acid probes specific for mRNA whichencodes H400.

I. Production of Mature H400

H400 can be produced by expressing a nucleotide sequence encoding it ina suitable expression system. Possible types of host cells include, butare not limited to, bacterial, yeast, insect, mammalian, and the like.Selecting an expression system, and optimizing protein productionthereby, involves the consideration and balancing of many factors,including (1) the nature of the protein to be expressed, e.g. theprotein may be poisonous to some host organisms, it may be susceptibleto degradation by host proteases, or it may be expressed in inactiveconformations or in insoluble form in some hosts, (2) the nature of themessenger RNA (mRNA) corresponding to the protein of interest, e.g. themRNA may have sequences particularly susceptible to host endonucleases,which drastically reduce the functional lifetime of the mRNA, or themRNA may form secondary structures that mask the start codon or ribosomebinding site, thereby inhibiting translation initiation in some hosts,(3) the selection, availability, and arrangement of host-compatibleexpression control sequences in the 3' and 5' regions flanking thecoding region--these include promoters, 5' and 3' protector sequences,ribosome binding sites, transcription terminators, enhancers,polyadenylate addition sites, cap sites, intron-splice sites, and thelike, (4) whether the protein has a secretion signal sequence which canbe processed by the host, or whether an expression control sequenceencoding a signal sequence endogenous to the host must be spliced ontothe region encoding the mature protein, (5) the available modes andefficiencies of transfection or transformation of the host, and whethertransient or stable expression is desired, (6) the scale and cost of thehost culture system desired for expressing the protein, (7) whether, andwhat type of, posttranslational modifications are desired, e.g. theextent and kind of glycosylation desired may affect the choice of host,(8) the ease with which the expressed protein can be separated fromproteins and other materials of the host cells and/or culture mediume.g. in some cases it may be desirable to express a fusion protein witha specialized signal sequence to aid in later purification steps, e.g.Sassenfeld et al., Biotechnology, January 1984, (9) the stability andcopy number of a particular vector in a selected host, e.g. Hofschneideret al., eds. Gene Cloning in Organisms Other than E. Coli (SpringerVerlag, Berlin, 1982), and (10) like factors known to those skilled inthe art.

Many reviews are available which provide guidance for making choicesand/or modifications of specific expression systems in light of therecited factors, e.g. to name a few, de Boer and Shepard, "Strategiesfor Optimizing Foreign Gene Expression in Escherichia coli," pgs.205-247, in Kroon, ed. Genes: Structure and Expression (John Wiley &Sons, New York, 1983), review several E. coli expression systems;Kucherlapati et al., Critical Reviews in Biochemistry, Vol. 16, Issue 4,pgs. 349-379 (1984), and Banerji et al., Genetic Engineering, Vol. 5,pgs. 19-31 (1983) review methods for transfecting and transformingmammalian cells; Reznikoff and Gold, eds., Maximizing Gene Expression(Butterworths, Boston, 1986) review selected topics in gene expressionin E. coli, yeast, and mammalian cells; and Thilly, Mammalian CellTechnology (Butterworths, Boston, 1986) reviews mammalian expressionsystems.

Likewise, many reviews are available which describe techniques andconditions for linking and/or manipulating specific cDNAs and expressioncontrol sequences to create and/or modify expression vectors suitablefor use with the present invention, e.g. Maniatis et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y.,1982); Glover, DNA Cloning: A Practical Approach, Vol. I and II (IRLPress, Oxford, 1985), and Perbal, A Practical Guide to Molecular Cloning(John Wiley & Sons, N.Y., 1984), to name only a few. Generally, withinan expression vector various sites may be selected for insertion of thecDNA of the invention. These sites are usually designated by therestriction endonuclease which cuts them and are well recognized bythose of skill in the art. Various methods for inserting DNA sequencesinto these sites to form recombinant DNA molecules are also well known.These include, for example, dG-dC or dA-dT tailing, direct ligation,synthetic linkers, exonuclease and polymerase-linked repair reactionsfollowed by ligation, or extension of the DNA strand with DNA polymeraseand an appropriate single-stranded template followed by ligation.

Often a vector containing the cDNA of the invention must be obtained inlarge quantities before transfection and/or transformation of cells in ahost culture can take place. For this purpose the vector is oftenreplicated without significant expression in an organism (the cloninghost) other than the one finally used for expression. In such cases,after propagation, the vectors are separated from the cloning host usingstandard techniques, e.g. as disclosed in Maniatis et al. (cited above).

Preferably H400 is produced by expressing the H400 cDNA carried bypcD(SRα)-H400 in a host cell suitable for pcD vectors, e.g. COS monkeycells (available from the ATCC under accession numbers CRL 1650 orCRL-1651), C127 mouse mammary tumor cell (available from ATCC underaccession number CRL 1616), or mouse L cells. Eukaryotic hosts arecapable of cleaving the signal peptide from the freshly translated H400polypeptide to form the mature H 400 polypeptide. Eukaryotic hosts arealso capable of producing other posttranslation modifications, such asglycosylation, which may be antigenically important.

Production of H400 in a bacterial expression system requires that thecleavage site of the signal peptide be determined, either for directexpression of a nucleic acid encoding the mature H400 polypeptide or forlinking such as nucleic acid to a bacterial sequence encoding anendogenous signal peptide. Empirical rules have been developed formaking such predictions with a high degree of accuracy, e.g. von Heijne,Eur. J. Biochem., Vol. 133, pgs. 17-21 (1983); J. Mol. Biol., Vol. 184,pgs. 99-105 (1985); Nucleic Acids Research, Vol. 14, pgs. 4683-4690(1986); and Perlman et al., J. Mol. Biol., Vol. 167, pgs. 391-409(1983). von Heijne's rules indicate that mature H400 begins with thealanine of position 23 with respect to the N-terminal amino acid ofFormula I (the predicted mature sequence is illustrated below in FormulaIII).

Mature H400 is produced as follows by COS7 cells transiently transfectedwith pcD(SRα)-H400. One day prior to transfection, approximately 10⁶ COS7 monkey cells are seeded onto individual 100 mm plates in Dulbecco'smodified Eagle medium (DME) containing 10% fetal calf serum and 2 mMglutamine. To perform the transfection, the medium is aspirated fromeach plate and replaced with 4 ml of DME containing 50 mM Tris.HCl pH7.4, 400 μg/ml DEAE-Dextran and 50 μg of the plasmid DNAs to be tested.The plates are incubated for four hours at 37° C., then theDNA-containing medium is removed, and the plates are washed twice with 5ml of serum-free DME. DME is added back to the plates which are thenincubated for an additional 3 hrs at 37° C. The plates are washed oncewith DME and then DME containing 4% fetal calf serum, 2 mM glutamine,penicillin and streptomycin is added. The cells are then incubated for72 hrs at 37° C. The growth medium is collected and H400 purifiedtherefrom using standard methods.

II. Immunogenic Peptides of H400

The present invention includes peptides derived from mature H400 whichform immunogens when conjugated with a carrier. The term immunogen asused herein refers to a substance which is capable of causing an immuneresponse. The term carrier as used herein refers to any substance whichwhen chemically conjugated to a peptide of the invention permits a hostorganism immunized with the resulting conjugate to generate antibodiesspecific for the conjugated peptide. Carriers include red blood cells,bacteriophages, proteins, or synthetic particles such as agarose beads.Preferably carriers are proteins, such as serum albumin, gamma-globulin,keyhole limpet hemocyanin, thyroglobulin, ovalbumin, fibrinogen, or thelike.

Peptides of the invention are defined in terms of their positions withinthe following amino acid sequence of mature H400: ##STR3##

Amino acids of Formula III are numbered with respect to the N-terminusof the mature H400 polypeptide. Thus, Ser₅ is serine at position 5.Peptides are defined as fragments of the mature H400 polypeptide. Thus,the peptide designated herein by Ser₅ -Cys₁₁ is equivalent to thepeptide Ser-Asp-Pro-Thr-Ala-Cys. Groups of peptides of the invention arealso defined in terms of fragments of the mature H400 polypeptide. Thus,the group designated herein as [Ser₅ -Cys₁₁ ]₅₋₆ consists of all 5 and 6amino acid peptides (i.e. all 5-mers and 6-mers) whose sequences areidentical to all or part of the peptide Ser₅ -Cys₁₁. That is, the groupconsists of the following five peptides: Ser-Asp-Pro-Pro-Thr-Ala,Asp-Pro-Pro-Thr-Ala-Cys, Ser-Asp-Pro-Pro-Thr, Asp-Pro-Pro-Thr-Ala, andPro-Pro-Thr-Ala-Cys.

Generally the invention includes all 6-mer to 24-mer peptide fragmentsof mature H400, i.e. [Ala₁ -Glu₆₆ ]₆₋₂₄. Preferably, the inventionincludes 6-mer to 24-mer peptide fragments which include the N-terminalor C-terminal sequences of the mature H400 polypeptide, or whichcorrespond to sequences of the mature H400 polypeptide that have highaverage hydrophilicity, as determined by Hopp-Woods or related analysis,e.g. Hopp and Woods, Proc. Natl. Acad. Sci., Vol. 78, pgs. 3824-3828(1981); or Kyte and Doolittle, J. Mol. Biol., Vol. 157, pgs. 105-132(1982). The latter set of preferred peptides of the invention includesthe following groups: [Ala₁ -Ser₂₀ ]₆₋₂₀ and [Thr43-Glu₆₆ ]₆₋₂₄.

Standard symbols are used throughout for amino acids, e.g. Cohen,"Nomenclature and Symbolism of alpha-Amino Acids," Methods inEnzymology, Vol. 106, pgs. 3-17 (Academic Press, N.Y., 1984).Accordingly, Table I of this reference is incorporated by reference.

Peptides of the invention are synthesized by standard techniques, e.g.Stewart and Young, Solid Phase Peptide Synthesis, 2 nd Ed. (PierceChemical Company, Rockford, IL, 1984). Preferably a commercial automatedsynthesizer is used, e.g. Vega Biochemicals (Tucson, AZ) models 296A orB, or Applied Biosystems, Inc. (Foster City, CA) model 430A.

Peptides of the invention are assembled by solid phase synthesis on across-linked polystyrene support starting from the carboxyl terminalresidue and adding amino acids in a stepwise fashion until the entire 34residue chain had been formed. The synthesis was performed on a fullyautomated peptide synthesizer (Applied Biosystems, Inc. model 430A). Thefollowing references are guides to the chemistry employed duringsynthesis: Merrifield, J. Amer. Chem. Soc., Vol. 85, pg. 2149 (1963);Kent et al., pg 185, in Peptides 1984, Ragnarsson, Ed. (Almquist andWeksell, Stockholm, 1984); Kent et al., pg. 217 in Peptide Chemistry 84,Izumiya, Ed. (Protein Research Foundation, B. H. Osaka, 1985);Merrifield, Science, Vol. 232, pgs. 341-347 (1986); and references citedin this latter reference.

In solid state synthesis it is most important to eliminate synthesisby-products, which are primarily termination, deletion, or modificationpeptides. Most side reactions can be eliminated or minimized by use ofclean, well characterized resins, clean amino acid derivatives, cleansolvents, and the selection of proper coupling and cleavage methods andreaction conditions, e.g. Barany and Merrifield, The Peptides, Cross andMeienhofer, Eds., Vol. 2, pgs 1-284 (Academic Press, N.Y., 1979). It isimportant to monitor coupling reactions to determine that they proceedto completion so that deletion peptides missing one or more residueswill be avoided. The quantitative ninhydrin reaction is useful for thatpurpose, Sarin et al. Anal. Biochem, Vol. 117, pg 147 (1981).Nα-t-butyloxycarbonyl (t-Boc)-amino acids were used with appropriateside chain protecting groups stable to the conditions of chain assemblybut labile to strong acids. After assembly of the protected peptidechain, the protecting groups were removed and the peptide anchoring bondwas cleaved by the use of low then high concentrations of anhydroushydrogen fluoride in the presence of a thioester scavenger, Tam et al.,J. Amer. Chem. Soc., Vol. 105, pg. 6442 (1983).

Side chain protecting groups used were Asp(OBzl), Glu(OBzl), Ser(Bzl),Thr(Bzl), Lys(Cl-Z), Tyr(Br-Z),

Arg(N^(G) Tos), Cys(4-MeBzl), and His(ImDNP). (Bzl, benzyl; Tos toluenesulfoxyl; DNP, dinitrophenyl; Im, imidazole; Z, benzyloxycarbonyl. Theremaining amino acids had no side chain protecting groups. All the aminoacids were obtained from Peninsula Laboratories, except thetBoc-His(ImDNP), which was from Chemical Dynamics and was crystallizedfrom ethanol before use. For each cycle the tBoc Nα protectedpeptide-resin was exposed to 65 percent trifluoroacetic acid (fromEastman Kodak) (distilled before use) in dichloromethane (DCM),(Mallenckrodt): first for 1 minute then for 13 minutes to remove theNα-protecting group. The peptide-resin was washed in DCM, neutralizedtwice with 10 percent diisopropylethylamine (DIEA) (Aldrich) indimethylformamide (DMF) (Applied Biosystems), for 1 minute each.Neutralization was followed by washing with DMF. Coupling was performedwith the preformed symmetric anhydride of the amino acid in DMF for 16minutes. The preformed symmetric anhydride was prepared on thesynthesizer by dissolving 2 mmol of amino acid in 6 ml of DCM and adding1 mmol of dicyclohexycarbodiimide (Aldrich) in 2 ml of DCM. After 5minutes, the activated amino acid was transferred to a separate vesseland the DCM was evaporated by purging with a continuous stream ofnitrogen gas. The DCM was replaced by DMF (6 ml total) at various stagesduring the purging. After the first coupling, the peptide-resin waswashed with DCM, 10 percent DIEA in DCM, and then with DCM. Forrecoupling, the same amino acid and the activating agent,dicyclohexylcarbodiimide, were transferred sequentially to the reactionvessel. After activation in situ and coupling for 10 minutes, sufficientDMF was added to make a 50 percent DMF-DCM mixture, and the coupling wascontinued for 15 minutes. Arginine was coupled as a preformedhydroxybenzotriazole (Aldrich) ester in DMF for 60 minutes and thenrecoupled in the same manner as the other amino acids. Asparagine andglutamine were coupled twice as preformed hydroxybenzotriazole esters inDMF, 40 minutes for each coupling. For all residues, the resin waswashed after the second coupling and a sample was automatically takenfor monitoring residual uncoupled α-amine by quantitative ninhydrinreaction, Sarin et al. (cited above).

The general technique of linking synthetic peptides to a carrier isdescribed in several references, e.g. Walter and Doolittle, "AntibodiesAgainst Synthetic Peptides," in Setlow et al., eds., GeneticEngineering, Vol. 5, pgs. 61-91 (Plenum Press, N.Y., 1983); Green et al.Cell, Vol. 28, pgs. 477-487 (1982); Lerner et al., Proc. Natl. Acad.Sci., Vol. 78, pgs. 3403-3407 (1981); Shimizu et al., U.S. Pat. No.4,474,754; and Ganfield et al., U.S. Pat. No. 4,311,639. Accordingly,these references are incorporated by reference. Also, techniquesemployed to link haptens to carriers are essentially the same as theabove-referenced techniques, e.g. chapter 20 in Tijsseu Practice andTheory of Enzyme Immunoassays (Elsevier, New York, 1985).

The four most commonly used schemes for attaching a peptide to a carrierare (1 ) glutaraldehyde for amino coupling, e.g. as disclosed by Kaganand Glick, in Jaffe and Behrman, eds. Methods of HormoneRadioimmunoassay, pgs. 328-329 (Academic Press, N.Y., 1979), and Walteret al. Proc. Natl. Acad. Sci., Vol. 77, pgs. 5197-5200 (1980); (2)water-soluble carbodiimides for carboxyl to amino coupling, e.g. asdisclosed by Hoare et al., J. Biol. Chem., Vol. 242, pgs. 2447-2453(1967); (3) bis-diazobenzidine (BDB) for tyrosine to tyrosine sidechaincoupling, e.g. as disclosed by Bassiri et al., pgs. 46-47, in Jaffe andBehrman, eds. (cited above), and Walter et al. (cited above); and (4)maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) for coupling cysteine(or other sulfhydryls) to amino groups, e.g. as disclosed by Kitagawa etal., J. Biochem. (Tokyo), Vol. 79, pgs. 233-239 (1976), and Lerner etal. (cited above).

A general rule for selecting an appropriate method for coupling a givenpeptide to a protein carrier can be stated as follows: the groupinvolved in attachment should occur only once in the sequence,preferably at the appropriate end of the segment. For example, BDBshould not be used if a tyrosine residue occurs in the main part of asequence chosen for its potentially antigenic character. Similarly,centrally located lysines rule out the glutaraldehyde method, and theoccurrences of aspartic and glutamic acids frequently exclude thecarbodiimide approach. On the other hand, suitable residues can bepositioned at either end of chosen sequence segment as attachment sites,whether or not they occur in the "native" protein sequence.

Internal segments, unlike the amino and carboxy termini, will differsignificantly at the "unattached end" from the same sequence as it isfound in the native protein where the polypeptide backbone iscontinuous. The problem can be remedied, to a degree, by acetylating theα-amino group and then attaching the peptide by way of its carboxyterminus.

The coupling efficiency to the carrier protein is conveniently measuredby using a radioactively labeled peptide, prepared either by using aradioactive amino acid for one step of the synthesis or by labeling thecompleted peptide by the iodination of a tyrosine residue. The presenceof tyrosine in the peptide also allows one to set up a sensitiveradioimmune assay, if desirable. Therefore, tyrosine can be introducedas a terminal residue if it is not part of the peptide sequence definedby the native H400 polypeptide.

Preferred carriers are proteins, and preferred protein carriers includebovine serum albumin, myoglobulin, ovalbumin (OVA), keyhole limpethemocyanin (KLH), or the like.

Peptides can be linked to KLH through cysteines by MBS as disclosed byLiu et al., Biochemistry, Vol. 18, pgs. 690-697 (1979). The peptides aredissolved in phosphate-buffered saline (pH 7.5), 0.1M sodium boratebuffer (pH 9.0) or 1.0M sodium acetate buffer (pH 4.0). The pH for thedissolution of the peptide is chosen to optimize peptide solubility. Thecontent of free cysteine for soluble peptides is determined by Ellman'smethod, Ellman, Arch. Biochem. Biophys., Vol. 82, pg. 7077 (1959).

For each peptide, 4 mg KLH in 0.25 ml of 10 mM sodium phosphate buffer(pH 7.2) is reacted with 0.7 mg MBS (dissolved in dimethyl formamide)and stirred for 30 min at room temperature. The MBS is added dropwise toensure that the local concentration of formamide is not too high, as KLHis insoluble in ≧30% formamide. The reaction product, KLH-MB, is thenpassed through Sephadex G-25 equilibrated with 50 mM sodium phosphatebuffer (pH 6.0) to remove free MBS, KLH recovery from peak fractions ofthe column eluate (monitored by OD₂₈₀) is estimated to be approximately80%.

KLH-MB is then reacted with 5 mg peptide dissolved in 1 ml of the chosenbuffer. The pH is adjusted to 7-7.5 and the reaction is stirred for 3 hrat room temperature. Coupling efficiency is monitored with radioactivepeptide by dialysis of a sample of the conjugate againstphosphate-buffered saline, and ranged from 8% to 60%.

III. Monoclonal Antibodies and Immunochemical Assays

Monoclonal antibodies are produced against mature H400 orpeptide-carrier conjugates by standard techniques, e.g. as disclosed byCampbell Monoclonal Antibody Technology (Elsevier, N.Y., 1984); Hurrell,ed. Monoclonal Hybridoma Antibodies: Techniques and Applications (CRCPress, Boca Raton, Fla., 1982); Schreier et al. Hybridoma Techniques(Cold Spring Harbor Laboratory, New York, 1980); U.S. Pat. No.4,562,003; or the like. In particular, U.S. Pat. No. 4,562,003 isincorporated by reference. For monoclonal antibody production, the firststep is to immunize a host animal to obtain a source of B lymphocytes.The B lymphocytes are fused with an appropriate immortalizing cell lineto form monoclonal antibody secreting hybridomas. Immortalizing celllines are usually tumor cell lines, such as myelomas. Preferably, thehost animals are rodents, and the immortalizing cell line is derivedfrom rodent cells. More preferably they are from the same species. Afterformation, hybridomas are screened for those producing antibodiesagainst the peptide of the invention. Immunization, lymphocyteharvesting, and cell fusion are all technique well known in the art.Roughly, immunization is carried out by a regimen of repeated injectionsinto the host animal of the purified peptide-carrier conjugate, usuallymixed with a suitable adjuvant. Immunization can be optimized by varyingseveral factors, including the amount of antigen used for the primaryinjection and subsequent boosts, the route of injection, the timeschedule for injecting and bleeding, and the use of adjuvant, e.g.Freund's complete or incomplete adjuvant. Techniques for fusion are alsowell known in the art, and in general, involve mixing the cells with afusing agent such as, most commonly, polyethylene glycol.

Successful hybridoma formation is assessed and selected by standardprocedures such as, for example, HAT selection. From among successfulhybridomas, those successfully secreting the desired antibody areselected by assaying the culture medium for their presence. Ordinarilythis is done using immunoreaction based assays, including, withoutlimitation, Western blot, ELISA, or RIA assays. The antibodies can berecovered from the medium using standard protein purificationtechniques.

"Two site" or "sandwich" immunoassays are the preferred immunoassays ofthe invention, e.g. as disclosed in U.S. Pat. No. 4,486,530.Accordingly, this patent is incorporated by reference. Such assaysentail the use of two different sets of anti-H400 antibodies, at leastone of which consists of a monoclonal antibody of the invention.Antibodies from one of the two sets are attached to the surface of asolid phase support. The attached antibodies are then exposed to asample suspected of containing H400. The H400 molecules bind to theattached antibodies. Next, the second set of antibodies is applied tothe bound H400, and binds to one or more antigenic determinants distinctfrom that (or those) to which the first set of antibodies is (or are)bound. The H400 is then detected by an indirect or direct signalgenerating means associated with the second set of antibodies. Forexample, the antibodies can be directly conjugated to a signalgenerating moiety, such as an enzyme, rare earth chelator, or an organicdye. Or, they can be indirectly linked to one or more signal generatingmoieties via additional antibodies, or high affinity complexes, such asthe avidin-biotin complexes. Quantitative measures of H400 concentrationare made by comparing the signal generated by the sample to signalsgenerated by H400 standards containing known concentrations of H400.

The invention includes kits of reagents for use in immunoassays,particularly sandwich immunoassays. Such kits include (1) a solid phasesupport, (2) a first antibody which is monoclonal and which is capableof binding to a first antigenic determinant of H400, (3) a secondantibody selected from the group consisting of a monoclonal antibodycapable of binding to a second antigenic determinant of H400 and apolyclonal antibody specific for H400 (referred to herein as a"polyclonal antibody composition"), and (4) a signal generation meansassociated with one of the three antibodies. Depending on the particularembodiment, kits may include a selection of two of the three anti-H400antibody types, either a monoclonal antibody specific for a firstantigenic determinant and a monoclonal antibody specific for a secondantigenic determinant, or a monoclonal antibody specific for a first orsecond antigenic determinant and a polyclonal antibody composition. Theantibodies may be in solution or in lyophilized form. One of the sets ofantibodies may come pre-attached to the solid support, or may be appliedto the surface of the solid support when the kit is used. The signalgenerating means may come pre-associated with one of the two antibodytypes, or may require combination with one or more components, e.g.buffers, antibody-enzyme conjugates, enzyme substrates, or the like,prior to use. Many types of signal generating means are available andcould make up one or more components of a kit. Various signal generatingmeans are disclosed by Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985). Kits of the invention may alsoinclude additional reagents, e.g. blocking reagents for reducingnonspecific binding to the solid phase surface, washing reagents, enzymesubstrates, and the like. The solid phase surface may be in the form ofmicrotiter plates, microspheres, or the like, composed of polyvinylchloride, polystyrene, or the like materials suitable for immobilizingproteins. Such materials having solid phase surfaces are referred toherein as "support means". Preferably, an enzyme which catalyzes theformation of a fluorescent or colored product is a component of thesignal generating means. More preferably, the enzyme is selected fromthe group consisting of peroxidase, alkaline phosphatase, andbeta-galactosidase. Substrates and reaction conditions for these enzymesare well known in the art, e.g. Tijssen (cited above).

IV. Nucleic Acid Probes

Nucleic Acid probes of the invention are constructed from nucleotidesequences determined by the nucleic acid of Formula II. The nature ofthe probe and its associated nucleic acid can vary depending on theparticular hybridization assay employed. One method of measuring H400mRNA is by cytoplasmic dot hybridization as described by White et al.,J. Biol. Chem., Vol. 257, pgs. 8569-8572 (1982) and Gillespie et al.,U.S. Pat. No. 4,483,920. Accordingly, these references are incorporatedby reference. Other approaches include in situ hybridization, e.g.Angerer et al., Genetic Engineering, Vol. 7, pgs. 43-65 (1985), and dotblotting using purified RNA, e.g. chapter 6, in Hames et al., eds.,Nucleic Acid Hybridization A Practical Approach (IRL Press, Washington,D.C., 1985). Generally, cytoplasmic dot hybridization involves anchoringmRNA from a cell or tissue sample onto a solid phase support, e.g.nitrocellulose, hybridizing a DNA probe to the anchored mRNA, andremoving probe sequences nonspecifically bound to the solid phasesupport or forming mismatched hybrids with the mRNA so that only probesequences forming substantially perfect hybrids with target mRNAsremain. The amount of DNA probe remaining is a measure of the number oftarget mRNA anchored to the solid phase support. The amount of DNA proberemaining is determined by the signal generated by its label.

Several standard techniques are available for labeling single and doublestranded nucleic acid fragments. They include incorporation ofradioactive labels, e.g. Harper et al., Chromosoma, Vol. 83, pgs.431-439 (1984); direct attachment of fluorescent labels, e.g. Smith etal., Nucleic Acids Research, Vol. 13, pgs. 2399-2412 (1985), andConnolly et al., Nucleic Acids Research, Vol. 13, pgs. 4485-4502 (1985);and various chemical modifications of the nucleic acid fragments thatrender them detectable immunochemically or by other affinity reactions,e.g. Tchen et al., Proc. Natl. Acad. Sci., Vol. 81, pgs. 3466-3470(1984); Richardson et al., Nucleic Acids Research, Vol. 11, pgs.6167-6184 (1983); Langer et al., Proc. Natl. Acad. Sci., Vol. 78, pgs.6633-6637 (1981); Brigati et al., Virology, Vol. 126, pgs. 32-50 (1983);Broker et al., Nucleic Acids Research, Vol. 5, pgs. 363-384 (1978); andBayer et al., Methods of Biochemical Analysis, Vol. 26, pgs. 1-45(1980).

Preferably probes are prepared by nick translation of a fragment ofpcD(SRα)-H400 containing all or the major portion of the H400 codingregion. For example, pcD(SRα)-H400 is amplified in JM101, isolated byethidium bromide cesium chloride density gradient equilibriumcentrifugation, digested with BamHI, and the restriction fragmentcarrying the entire coding region for H400 is separated by gelelectrophoresis. The fragment containing the coding region (which isreadily identified by a size marker) is extracted from the gel and nicktranslated in the presence of one or more kinds of labeled nucleotides,e.g. Maniatis et al., Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory, New York, 1982); or Brigati et al. (citedabove). After removal of unincorporated nucleotides, the probe isapplied to the anchored mRNA at a concentration in the range of betweenabout 1-50 ng/ml (with a specific activity in the range of about 1-2×10⁸cpm/μg probe).

Peripheral blood lymphocytes (PBLs) (which contain about 80% T cells)are used as a source of T cells for the assay. PBLs are obtained bystandard techniques, e.g. Boyum, Scand. J. Clin. Lab. Invest., Vol. 21(Suppl. 97), pg. 77 (1968). If desired, a fraction of cells enriched inT cells (to about 95%) can be obtained using standard techniques, e.g.eliminating B cells by rosetting, Gmelig-Meyling et al., Vox Sang., Vol.33, pg. 5 (1977). Generally, PBLs are obtained from fresh blood byFicoll-Hypaque density gradient centrifugation. Preferably mRNA fromPBLs is anchored for hybridization to the probe by the followingprotocol. Isolated PBLs are lysed by suspending in a lysis buffer (0.14MNaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl pH 8.6 , 0.5% Nonidet P-40 (anonionic detergent, e.g. from Sigma)) at 4° C. at a final concentrationof 1×10⁸ cells/ml. The suspension is vortexed for 10 sec and the nucleiare pelleted (13,000 g, 2.5 min). The resulting cytoplasmic lysates arethen transferred to a sterile 1.5 ml tube containing 0.3 volumes of 20×SSC (1× SSC=0.15M NaCl, 0.015M trisodium citrate (standard salinecitrate)) and 0.2 volumes of 37% (w/w) formaldehyde. The mixture is thenincubated at 60° C. for 15 min and stored in aliquots at -70° C. Foranalysis, 15 μl of each sample is titered by serial three fold dilutionsin 15× SSC into a 96-well flat-bottomed microtiter plate (Falcon, BectonDickinson, Oxnard, CA) in a 0.1 ml. Each dilution is applied withsuction to a sheet of Nytran (a modified nylon support available fromSchleicher and Schuell, Keene, NH; 0.45 μm pore size) supported on afilter paper (Whatman 3 mmChr, Whatman Inc., Clifton, N. J.) utilizing a96 hold Minifold apparatus (Schleicher and Schuell). The Nytran paper isthen baked (80° C., 2 H) and treated with a prehybridization solutionconsisting of 50% formamide (BRL, Gaithersburg, MD) 6× SSC, 50 μg/ml E.coli tRNA (Sigma), 0.2% (w/v) each of ficoll (M_(w) =400,000),polyvinylpyrollidone, and bovine serum albumin (BSA). The probe isapplied to the Nytran support at a concentrate of about 50 ng probe/mlof prehybridization solution. Following hybridization, the support iswashed two times for 15 min each at room temperature in 2× SSC, thentwice for 30 min each at 60° C. in 2× SSC/0.5% SDS. The support is thenexposed to film using an intensifying screen and quantitated by scanningwith a laser densitometer (e.g. Ultroscan XL, LKB Instruments Inc.,Gaithersburg, MD).

If cytoplasmic dot hybridization lacks sufficient sensitivity,preferably the RNA is first extracted from the PBLs prior to blotting.For example, RNA may be extracted by the guanidinium thiocyanate methoddisclosed by Chirgwin et al., in Biochemistry, Vol. 18, pgs. 5294-5299(1979).

The descriptions of the foregoing embodiments of the invention have beenpresented for the purpose of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formsdisclosed, and obviously many modifications and variations are possiblein light of the above teaching. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical application to thereby enable others skilled in the art tobest utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto.

Applicants have deposited E. coli JM101 carrying pcD(SRα)-H400 with theAmerican Type Culture Collection, Rockville, MD, USA (ATCC), underaccession number 67614. This deposit was made under conditions asprovided under ATCC's agreement for Culture Deposit for Patent Purposes,which assures that the deposit will be made available to the U. S.Commissioner of Patents and Trademarks pursuant to 35 USC 122 and 37 CFR1.14, and will be made available to the public upon issue of a U.S.patent, which requires that the deposit be maintained. Availability ofthe deposited strain is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

We claim:
 1. A nucleic acid capable of encoding the mature polypeptideof the open reading frame defined by the following amino acid sequence:##STR4##
 2. The nucleic acid of claim 1 defined by the followingnucleotide sequence: ##STR5##